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Molecular Project: Polymerase Chain Reaction Essay


A transposable element (TE), which is also known as a transposon, is a sequence of DNA that is capable of altering its position within a genome (Dalby, Pereira, & Goldstein, 1995). Consequently, it can generate or undo transmutations leading to a change in the size of the genome. Transposons serve a useful role in the evolution and working of the genome. Two forms of transposons exist: autonomous and non-autonomous. Autonomous transposons move on their own while non-autonomous TEs can only move in the presence of another transposon because of the lack of transposase enzyme in Class II transposons or reverse transcriptase in Class I transposons (Robillard, Le Rouzic, Zhang, Capy, & Hua-Van, 2016). A native P element is a transposable element specific to the fruit fly Drosophila melanogaster. Its main role in scientific studies is to allow the performance of mutagenesis and the development of genetically modified flies used for hereditary investigations.

The native P element is surrounded by terminal inverted repeats that aid in its movement. The full length of a native P element is approximately 2.9 kb. It encodes a transposase protein whose size is 87 kDa. The synthesis of this protein is confined to the germline by a controlled, tissue-specific pre-mRNA joining machinery. Native P elements move through “cut-and-paste” machinery and form an eight base-pair target site duplication on insertion (Mourier, 2016). The activity of the P-element transposase requires GTP and magnesium as cofactors, which make it unique. Also, approximately 150 base pairs of DNA sequence are required at either end of the P element for its transposition. The composition of these sequences includes 31-bp end reversed repeats, interior transposase-binding positions, and inner 11-bp reversed replications.

Engineered P elements or the EP elements have an origin of replication as well as selectable markers to facilitate the identification of entities in which the transposition events have occurred (Dalby et al., 1995). P elements with deletions in the transposase encoding segment are assembled when transposase action is provided in trans in one cohort. EP elements move about the genome by the use of transposase.

The transgenic lines we are working with are the wild-type and the white lines. These lines are identified and monitored by the description of the phenotype expressed in the fruit fly. The white gene controls eye coloring in Drosophila. When P element vectors with the mini-white gene devoid of enhancers are inserted into arbitrary chromosomal regions, transformed flies with the mini-white gene exhibit a variety of eye colors ranging from pale yellow to red. The actual color is determined by the site of mini-white addition into the genome.

This experiment aims to extract genomic DNA from Drosophila melanogaster and identify the location of the EP transposable element in the new fly line generated earlier. The crossing scheme for the classical genetics project involves digestion, circularization, and transformation of the Drosophila genome. Flies bearing the new allele generated in the scheme are identified by combining the mutagenized flies in pools and generating probes matching the sequences next to each site of insertion. These probes are then used for hybridization to cloned genomic intervals permitting the samples with the insertions to be distinguished.

PCR (polymerase chain reaction) is a molecular biology technique that utilizes enzymes to amplify or make copies of specified regions of DNA without the need to carry out cloning (Ochman, Gerber, & Hartl, 1988). PCR requires that the DNA sequences of the fragment of interest are known. DNA polymerase then uses short sequences of DNA from the fragment to be amplified as a template to extend the DNA. The general process of PCR entails heating the DNA fragments at 95oC for 90 seconds to denature the DNA and separate the double helix into single strands. The denatured DNA is cooled down at 48 oC for 60 seconds together with oligonucleotides primers, which are short DNA sequences corresponding to the 5’ and 3’ ends of the DNA fragment of interest, to anneal the primers to the template (Ochman et al., 1988).

Taq polymerase, a type of DNA polymerase that works at high temperatures, uses deoxynucleotides to build new strands of DNA using the original denatured DNA as a template. This process occurs at 70 oC for 4 minutes (Ochman et al., 1988). Consequently, the original double-stranded DNA is duplicated. The entire process of denaturation, annealing, and extension is repeated several times (about 30 times) such that with each cycle, a new double-stranded DNA consisting of an old and a new strand is created (Ochman et al., 1988). As a result, the original DNA duplicates, and both new molecules contain one old and one new strand of DNA. Currently, the PCR process is mechanized using a gadget known as a thermocycler. A thermocycler automatically regulates the temperature of the reactions at specified intervals for denaturation and the formation of new DNA strands.

Molecular studies and analyses often require the use of large amounts of DNA. However, obtaining large quantities of DNA may be impossible especially for genes that are expressed in small quantities in the cell. Therefore, PCR helps to increase the number of copies of such genes for further molecular analyses. The human genome project is an example of a project that has benefitted from PCR. In the medical setting, PCR is beneficial in the molecular diagnoses of diseases such as human immunodeficiency virus (HIV) and malaria parasites. The diagnosis of genetic disorders is also carried out using PCR. In forensic sciences, DNA fingerprinting, which helps to identify paternity or link a suspected criminal to a crime scene, has relied heavily on PCR. This technique is useful for this project because it enables the amplification of the sequences surrounding the EP element downstream and upstream. The amplified sequences are then sequenced for subsequent characterization of the consensus sequence of the insertion of transposable elements as well as the residual sequences following partial excision.

Inverse PCR is a modification of the conventional polymerase chain reaction. In this method, it is possible to carry out downstream or upstream amplification of DNA regions that are adjacent to a known sequence of DNA. Inverse PCR has been applied in molecular genetics in the amplification and identification of sequences adjacent to transposable elements. Inverse PCR is used to figure out the molecular location of the EP element in question by amplifying the locations surrounding the 5’ and 3’ ends of the EP element. As a result, it is possible to characterize the sequences surrounding the EP element.

An illustration of inverse PCR to amplify the regions flanking the 5’and 3’ ends of an EP transposon with mini-white markers.
Figure 1: An illustration of inverse PCR to amplify the regions flanking the 5’and 3’ ends of an EP transposon with mini-white markers.

The PE element is engineered to contain four MspI restriction sites with two sites flanking the mini-white marker. The entire sequence is digested with MspI restriction enzyme to yield three fragments: the EP element, the 5′ fragment, and the 3′ fragment. The regions flanking the EP element are then ligated to circularize them. Inverse PCR is then carried out with primers that identify the 5’ and 3’ ends of the element. The PCR products are then sequenced.

References

Dalby, B., Pereira, A. J., & Goldstein, L. S. (1995). An inverse PCR screen for the detection of P element insertions in cloned genomic intervals in Drosophila melanogaster. Genetics, 139(2), 757-766.

Mourier, T. (2016). Potential movement of transposable elements through DNA circularization. Current Genetics, 62(4), 697-700.

Ochman, H., Gerber, A. S., & Hartl, D. L. (1988). Genetic applications of an inverse polymerase chain reaction. Genetics, 120(3), 621-623.

Robillard, É., Le Rouzic, A., Zhang, Z., Capy, P., & Hua-Van, A. (2016). Experimental evolution reveals hyperparasitic interactions among transposable elements. Proceedings of the National Academy of Sciences, 113(51), 14763-14768.

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IvyPanda. 2020. "Molecular Project: Polymerase Chain Reaction." August 28, 2020. https://ivypanda.com/essays/molecular-project-polymerase-chain-reaction/.

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IvyPanda. (2020) 'Molecular Project: Polymerase Chain Reaction'. 28 August.

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